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Association of xanthine oxidase with the bovine milk-fat-globule membrane. Nature of the enzyme-membrane association.

ABSTRACT: 1. Xanthine oxidase (EC 1.2.3.2) was found to represent more than 8% of the intrinsic protein of the bovine milk-fat-globule membranes. 2. Less than 25% of the xanthine oxidase activity of the fat-globule membrane was solubilized with 0.1 M-sodium pyrophosphate buffer or 2M-NaCl. Of the particulate activity remaining 56% was solubilized with Triton X-100. 3. The xanthine oxidase activity solubilized with buffer, 2M-NaCl or Triton X-100 was not liberated as the free enzyme. Only tryptic digestion was found to release the free enzyme from the fat-globule membrane. Tryptic digestion also liberated free xanthine oxidase from those fractions solubilized by buffer or NaCl, but not from those fractions solubilized with Triton X-100 or by sonication. 4. The effect of membrane association on the catalytic properties of the enzyme could be mimicked by low pH or by the presence in the assay mixture of certain concentrations of 2-methyl-propan-2-ol, but not 1,4-dioxan, suggesting that hydrogen-bonding rather than low dielectric constant may be involved. 5. The origin of the milk-fat-globule membrane is discussed with reference to the intrinsic nature of the associated xanthine oxidase activity.

SUBMITTER: Briley MS

PROVIDER: S-EPMC1165467 | biostudies-other | 1975 Jun

REPOSITORIES: biostudies-other

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Association of xanthine oxidase with the bovine milk-fat-globule membrane.

Project description:1. The catalytic properties of xanthine oxidase in bovine milk (EC 1.2.3.2) are dependent on the state of the enzyme, i.e. whether free or bound to the fat-globule membrane. Oxidase activity of the membrane-bound enzyme towards NADH is enhanced relative to that towards xanthine. This reflects a change in the relative K(m) values and enables the ratio of xanthine to NADH oxidase activities (X/N) to be used as a parameter for the relative amounts of free and membrane-bound xanthine oxidase in milk fractions. 2. Chromatography of buttermilk on Sepharose 2B yielded an excluded fraction, BM(1), with xanthine oxidase activity. The remaining xanthine oxidase activity was eluted as a single broad peak. This was further resolved on Sephadex G-200 into an excluded fraction, BM(2), and free xanthine oxidase. Fractions BM(1) and BM(2) had X/N values in the range 45-65, which is characteristic of membrane-bound xanthine oxidase. Purified xanthine oxidase has a mean X/N value of 110.3. Addition of fraction BM(1), heated to remove associated enzyme activities, to purified xanthine oxidase progressively enhanced its NADH oxidase activity to a value where its X/N value was characteristic of membrane-bound xanthine oxidase. This was shown to be due to binding of free enzyme to heated fraction BM(1). The binding constant and stoicheiometry were determined. 4. Proteolytic digestion of fraction BM(1) liberated free xanthine oxidase from the fat-globule membrane with a corresponding alteration in X/N value.

| S-EPMC1168363 | biostudies-other

Preparation of monoclonal antibodies to xanthine oxidase and other proteins of bovine milk-fat-globule membrane.

Project description:Nine hybridomas secreting monoclonal antibody to proteins of bovine milk-fat-globule membrane were isolated. All nine cell lines continued to secrete monoclonal antibody after serial transfer in culture and after passage as solid tumours in Balb/cJ mice. Four of the cell lines secreted monoclonal antibody specific for xanthine oxidase, one of the major proteins of milk-fat-globule membrane.

| S-EPMC1161979 | biostudies-other

Xanthine Oxidoreductase mediates milk fat globule secretion.

Project description:Analysis of the milk fat globule proteome from mice with a mammary-specific knockout of Xanthine Oxidoreductase.

2017-05-31 | PXD003336 | Pride

Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: structure-based mechanism of conversion.

Project description:Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. Here, we present the crystal structure of the dimeric (M(r), 290,000) bovine milk XDH at 2.1-A resolution and XO at 2.5-A resolution and describe the major changes that occur on the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20-kDa domain containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423Lys 433). This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme.

| S-EPMC27090 | biostudies-literature

Bovine milk-fat-globule membrane contains an enzymically inactive form of acetyl-CoA carboxylase.

Project description:Enzymically inactive acetyl-CoA carboxylase [acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] was found as a component of bovine milk-fat-globule membrane (MFGM). Acetyl-CoA carboxylase was present in MFGM at a higher concentration than in cytosolic or mitochondrial fractions of bovine mammary tissue, which makes it unlikely that its presence was due to simple contamination by these subcellular constituents.

| S-EPMC1135677 | biostudies-other

Isolation and structural characterization of alkali-labile oligosaccharides from bovine milk-fat-globule membrane.

Project description:Treatment of sialoglycopeptides derived from bovine milk-fat-globule membrane with alkaline borohydride released a reduced oligosaccharide fraction from which a tetrasaccharide and two trisaccharides were isolated. Periodate-oxidation studies coupled with methylation analysis and g.l.c.-mass spectrometry established their structures as: N-acetylneuraminyl-(2 leads to 3)-beta-D-galactopyranosyl-(1 leads to 3)-[N-acetylneuraminyl-(2 leads to 6)]-N-acetyl-D-galactosaminitol, N-acetylneuraminyl-(2 leads to 3)-D-glactopyranosyl-(1 leads to 3)-N-acetyl-D-galactosaminitol and beta-D-galactopyranosyl-(1 leads to 3)-[N-acteylneuraminyl-(2 leads to 6)]-N-acetyl-D-galactosaminitol respectively.

| S-EPMC1183999 | biostudies-other

The isolation of demolybdo xanthine oxidase from bovine milk.

Project description:It was deduced many years ago from indirect evidence that demolybdo xanthine oxidase is present in normal bovine milk. This has now been confirmed by isolation of this enzyme form by a method based on the folate-gel affinity-chromatography procedure described Nishino & Tsushima [(1986) J. Biol. Chem. 261, 11242-11246]. Enzymic and spectroscopic properties of demolybdo xanthine oxidase, which retains flavin and iron-sulphur centres, are generally in accordance with expectations. Like the normal enzyme, it yields on denaturation material fluorescing at 460 nm. Molybdenum cofactor activity measured by the Neurospora crassa nit-1 assay in the presence of added molybdate was 33% of that of the normal enzyme. The absorption spectrum in the near-u.v. region differs slightly, but significantly, from that of the active and desulpho forms of the enzyme. It is concluded that the molybdenum cofactor site contains a pterin-like material not identical with that in the normal enzyme. The significance of the occurrence of demolybdo xanthine oxidase in milk is discussed, and evidence in the literature for demolybdo forms of other molybdoenzymes is briefly reviewed. Additional studies on the use of the affinity procedure for large-scale preparation of high-activity xanthine oxidase are described. In agreement with our ability to isolate the demolybdo enzyme, the procedure appears less effective in eliminating the demolybdo than the desulpho enzyme.

| S-EPMC1135333 | biostudies-other

Role of milk fat globule membrane (MFGM) in modulating gene expression in humans

Project description:The aim of this study was to investigate if milk fat globule membrane (MFGM) enclosing the dairy fat influence peripheral blood mononuclear cells (PBMC) gene expression. This study was a 8-week single-blind, randomized, controlled isocaloric trial with two parallel groups including overweight (mean BMI: 28) adult women (n=30). All subjects consumed 40 g dairy fat per day either as cream (MFGM diet) or as butter oil (control diet). MFGM diet and control diet showed differential responses in plasma cholesterol. Absolute differences in gene expression were calculated for each gene in each subject, comparing after with before the intervention. These absolute differences in gene expression were compared between intervention groups with T-test and with Significance Analysis of Microarrays (SAM). 19 genes were significantly different regulated by the two diets with a FDR of 23%. Most of thechanges in gene expression correlated with changes in blood lipids. Patients were examined in the morning after 12 h's fasting. Whole blood was obtained before (W0) and after completion (W8) of the dietary intervention for isolation of PBMCs and RNA extraction. From total RNA we prepared and hybridised biotinylated complementary RNA to GeneChip Human Gene 1.1 ST Arrays (Affymetrix Inc., Santa Clara, CA), and then washed, stained and scanned the slides using standardised protocols (Affymetrix Inc.). Signicance analysis of microarrays (SAM) was use to compare the difference in gene expression between groups.

2015-08-01 | E-GEOD-60148 | biostudies-arrayexpress

Quantitative Label-Free Proteomic Analysis of Milk Fat Globule Membrane in Donkey and Human Milk.

Project description:Previous studies have found donkey milk (DM) has the similar compositions with human milk (HM) and could be used as a potential hypoallergenic replacement diet for babies suffering from cow's milk allergy. Milk fat globule membrane (MFGM) proteins are involved in many biological functions, behaving as important indicators of the nutritional quality of milk. In this study, we used label-free proteomics to quantify the differentially expressed MFGM proteins (DEP) between DM (in 4-5 months of lactation) and HM (in 6-8 months of lactation). In total, 293 DEP were found in these two groups. Gene Ontology (GO) enrichment analysis revealed that the majority of DEP participated in regulation of immune system process, membrane invagination and lymphocyte activation. Several significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were determined for the DEP, such as lysosome, galactose metabolism and peroxisome proliferator-activated receptor (PPAR) signaling pathway. Our study may provide valuable information in the composition of MFGM proteins in DM and HM, and expand our knowledge of different biological functions between DM and HM.

| S-EPMC8258387 | biostudies-literature

Milk Fat Globule Membrane Supplementation in Children: Systematic Review with Meta-Analysis.

Project description:(1) Background: Milk fat globule membrane (MFGM), composing fat droplets responsible for lipid transport in breast milk, has been shown to possess immunological and antimicrobial effects. Standard formulas (SF) are devoid of MFGMs during the production process. The study's aim was to evaluate the safety and benefits of MFGMs supplementation in children. (2) Methods: We searched four databases for randomized controlled trials evaluating the supplementation of MFGMs in children. Growth parameters were chosen as the primary outcome. (3) Results: Twenty-four publications of seventeen studies were included. Meta-analyses assessing the primary outcomes at the age of 4 months included four studies (814 children) comparing the MFGM-supplemented formulas and SF, and two trials (549 children) comparing the MFGM-supplemented formulas and breastfeeding. The primary outcomes were non-inferior in all the experimental MFGM formulas compared to SF, or even represented more similar results to breastfed infants. The promising effects, including a lower incidence of acute otitis media and improved cognitive development, cannot be firmly confirmed due to the small amount of existing evidence. No significant adverse effects were reported in any of the assessed products. (4) Conclusions: The available data signaled beneficial effects and a good safety profile, requiring future research with well-designed trials.

| S-EPMC7996302 | biostudies-literature

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